Patients with cystic fibrosis (CF) suffer from chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa (PA), and experience worsening, irreversible lung injury leading to premature death. This injury is mediated by an inflammatory process that results, at least in part, from stimulation of the innate immune system by PA lipid A, the bioactive component of lipopolysaccharide (LPS). Preliminary data indicates that PA responds to the CF lung environment by synthesizing lipid A structures not present in clinical isolates from non- CF PA infections. This project will test the hypothesis that CF-specific lipid A structures promote resistance to innate immune mechanisms such as antimicrobial peptides and result in increased inflammatory responses. Key steps in the synthesis of CF-specific lipid A are hydroxylation, palmitoylation, and the synthesis and transfer of aminoarabinose. Genes encoding the relevant enzymes will be defined, and insertional mutants will be constructed and tested in assays of antimicrobial peptide resistance. Aerosolization of mutant whole bacteria or LPS into the murine airway or incubation in cell culture will be used to test the role of CF-specific lipid A modifications in inflammatory responses. CF-specific lipid A structures will be purified and their biological activity characterized. Finally, outer membrane proteins (OMPs) coordinately regulated with CF-specific lipid A will be identified by proteomic analysis, and the role of such OMPs in antimicrobial peptide resistance will be assayed in insertional mutants. These studies will provide insight into bacterial mechanisms that contribute to CF lung disease, including the role of lipid A modifying enzymes. Such enzymes may provide novel targets for the development of drugs to treat PA lung infections and their inflammatory consequences.